Abstract

Collagen peptides (CPs) have demonstrated to exert beneficial effects on skin photoaging. However, little has been done to evaluate their effects on chronologically aged skin. Here, the effects of CPs from bovine bone on skin aging were investigated in chronologically aged mice. 13-month-old female Kunming mice were administered with CPs from bovine bone (200, 400 and 800 mg/kg body weight/day) or proline (400 mg/kg body weight/day) for 8 weeks. Mice body weight, spleen index (SI) and thymus index (TI), degree of skin laxity (DSL), skin components, skin histology and antioxidant indicators were analyzed. Ingestion of CPs or proline had no effect on mice skin moisture and hyaluronic acid content, but it significantly improved the skin laxity, repaired collagen fibers, increased collagen content and normalized the ratio of type I to type III collagen in chronologically aged skin. CPs prepared by Alcalase performed better than CPs prepared by collagenase. Furthermore, CPs intake also significantly improved the antioxidative enzyme activities in skin. These results indicate that oral administration of CPs from bovine bone or proline can improve the laxity of chronologically aged skin by changing skin collagen quantitatively and qualitatively, and highlight their potential application as functional foods to combat skin aging in chronologically aged process.

Keywords: collagen peptides, bovine bone, proline, skin aging, chronologically aged mice, antioxidative enzymes

1. Introduction

The impact of aging on the appearance and function of skin has received increasing attention in recent decades. It is widely accepted that skin aging is distinguished into chronological skin aging and skin photoaging. Skin photoaging is caused by solar radiation and it is common in sunlight-exposed skin, especially in the face. Therefore, skin photoaging could be prevented or decreased by photo-protection. The common clinical signs of photoaged skin include deep and coarse wrinkles, dryness, sallowness and laxity. In contrast, chronological skin aging is caused by passage of time and it takes place all the time in whole-body skin, including facial skin. Chronologically aged skin is characterized by fine wrinkling and laxity. Chronological skin aging accounts for a great part of skin aging and it is more common than skin photoaging in dark skinned individuals and females. A youthful appearance is considered to play an important role in keeping self-esteem and social relations. Therefore, there is increasing demand for anti-aging interventions to delay or even reverse signs of skin aging.

The use of diet supplements to improve the appearance and function of aged skin has received growing attention. Many dietary components, such as polyphenols, vitamins, fatty acids, trace minerals and proteins, have reported to exert beneficial effects on aged skin and have been used as nutraceuticals or functional foods in many counties and regions. Recently, researchers have paid much attention to protein hydrolysates as potential dietary supplements. Collagen is the main structural protein of the different connective tissues, such as skin, bone, cartilage and tendons, and has been widely used in the medicine and food industries. Collagen peptides (CPs) are the enzymolysis product of collagen or gelatin and they are used as important active components because of their various bioactivities, high bioavailability and good biocompatibility. Several studies have demonstrated the beneficial effects of CPs ingestion on skin photoaging. Oral administration of CPs from fish skin had obvious protective effects on photoaging skin, including improving moisture retention ability, repairing the endogenous collagen and elastin protein fibers. In addition, clinical trials have also demonstrated that the beneficial effects of CPs intake on facial skin, including improving facial skin elasticity, reduce skin dryness and wrinkles, and increase the collagen content of the skin dermis. However, little work was performed to evaluate the effects of CPs intake on chronologically aged skin.

Bovine bone is the main by-products in the bovine processing industry and has been widely used as raw material to obtain high-quality gelatin. Although there are some concerns with mad cow disease in Europe and the United States, bovine bone is still one of the most abundant sources of gelatin and accounts for 23.1% of the gelatin production. Therefore, bovine bone is an abundant and high-quality raw material used to prepare CPs. The biological effect of CPs from bovine bone is mainly concentrated on its beneficial effect on bone metabolism, including inhibition of bone loss and improvement of osteoarthritis. However, there is limited knowledge about the effect of CPs from bovine bone on skin aging. Therefore, preparing CPs from bovine bone and further evaluating its effect on skin aging is a good way to utilize the by-products for an economical and environmental advantage.

The functional activities of protein-derived hydrolysates or peptides are greatly impacted by their molecular structure and weight, which are highly affected by their processing conditions and especially enzyme specificity. Alcalase is a common protease and widely used to prepare protein hydrolysate or peptides. It is a typical endoprotease and preferentially cleaves sites containing hydrophobic residues, such as Ala, Leu, Val and Phe. Bacterial collagenase is a protease that hydrolysates collagen. It has a preference for X-Gly (X is usually a neutral amino acid) bond of the -Gly-Pro-X-Gly-Pro-X- repeating sequence in the collagen molecule. Collagenase has a great promise in collagen processing industry. Considering enzyme specificity, the molecular structure or sequences of peptides produced by these two enzymes may be different, which may greatly impact their effects on chronologically aged skin.

The objective of the present study is to investigate the effects of CPs from bovine bone on skin aging based on the chronologically aged model. Bovine bone was employed as a raw material to prepare different CPs using Alcalase and collagenase. Then, the effects of CPs from bovine bone on chronologically aged skin were investigated in chronologically aged mice by analyzing the skin histology, skin components and antioxidative indicators. The results showed that oral administration of CPs from bovine bone has beneficial effects on chronologically aged skin by improving the skin laxity, but it had no on moisture retention of skin. CPs prepared by Alcalase performed better than CPs prepared by collagenase.

2. Materials and Methods

2.1. Materials and Chemicals

Alcalase was purchased from Novozymes (Beijing, China). Proline (food grade) and bacterial collagenase were purchased from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay kit was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Commercial kits used for determining hydroxyproline (Hyp), type I and type III collagen, hyaluronic acid (HA), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were purchased from Jiancheng Inst. of Biotechnology (Nanjing, China). All other chemicals used in the study were of analytical grade or better.

2.2. Collagen Peptides (CPs) Preparation
Gelatin was extracted from bovine bone with hot water. Briefly, the bovine bone was treated in boiling water for 6 h, followed by removing bone using gauze filter. The filtrate was cooled, defatted and centrifuged at 4500× g for 15 min with a refrigerated centrifuge (TGL-185, Pingfan Co., Ltd., Changsha, China). After centrifugation, the upper soluble fractions were collected and freeze-dried to obtain the gelatin. The gelatin was enzymatically hydrolyzed by the Alcalase at pH 8.0 for 4.0 h to obtain collagen peptides (named ACP), and the collagenase at pH 7.5 for 3.0 h to obtain collagen peptides (named CCP). Finally, the hydrolysates were dialyzed to discard salt and free amino acids, freeze-dried and stored at −80 °C until use.

2.3. Molecular Weight Distribution
The molecular weight distribution of CPs was measured using a Shimadzu LC-15C high performance liquid chromatography (HPLC) system (Shimadzu, Tokyo, Japan) equipped with a TSK gel G2000 SWXL column (7.8 × 300 mm, Tosoh, Tokyo, Japan). Samples were loaded onto the column and eluted with 45% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.5 mL/min and monitored at 214 nm at room temperature. A molecular weight calibration curve (y = −0.1881x + 6.5867, y: log MW, x: time, R2 = 0.9954) was obtained from the average retention times of the following standards: Gly–Ser (146 Da), Asn–Cys–Ser (322 Da), Trp–Pro–Trp–Trp (674 Da), bacitracin (1423 Da) and aprotinin (6512 Da).

2.4. Amino Acid Composition
The samples were hydrolyzed in 6.0 M HCl at 110 °C for 24 h. After phenylisothiocyanate (PITC) derivatization reaction, the amino acid composition was analyzed by a Shimadzu LC-15C high performance liquid chromatography (HPLC) system (Shimadzu, Tokyo, Japan) equipped with a reverse Zorbax SB-C18 column (4.6 × 250 mm, Agilent, Santa Clara, CA, USA). The mobile phase consisted of (A) 10 mM phosphate buffer solution (pH 6.9) and (B) 100% acetonitrile and the flow rate was 1.0 mL/min. The gradient was programmed as follows: 0–5 min, 5–10% B; 5–25 min, 10–17% B; 25–45 min, 17–35% B; 45–48 min, 35–100% B; 48–50 min, 100% B; 50–58 min, 100–5% B; and 58–60 min, 5% B. The detection wavelength was set at 254 nm.

2.5. Animals, Diets, and Treatments

Animal experiments were carried out under the protocols approved by the Committee for Animal Research of Peking University and followed the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1996). The present experiment was approved by the Animal Experimental Welfare & Ethical Inspection Committee, the Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture (Beijing, China), and was performed in the Experimental Animal Center, Supervision and Testing Center for GMOs Food Safety, Ministry of Agriculture (SPF grade, Beijing, China).

Two-month-old (young mice, 28 ± 2 g, specific pathogen free (SPF) grade) and thirteen-month-old (old mice, 45 ± 5 g, SPF grade) female Kunming mice were purchased from Sibeifu (Beijing) Laboratory Animal Science and Technology Co., Ltd. (Beijing, China). The two-month-old mice were set as young controls (n = 10) and were given 0.2 mL normal saline. The thirteen-month-old mice were divided, based on body weight, into 6 groups (n = 10/group), including the model group and CPs treatment groups. The model group was given 0.2 mL normal saline; whereas the CPs treatment groups were given 0.2 mL ACP at doses of 200 (ACP-200), 400 (ACP-400) and 800 mg/kg body weight (ACP-800), respectively, and 0.2 mL CCP at a dose of 400 mg/kg body weight (ACP-400). In addition to free access to normal AIN-93M purified diet and water, each group was intragastrically administrated with 0.2 mL of normal saline or CPs once a day for eight weeks. After eight weeks, mice were sacrificed and samples were collected for further treatment and analysis.

2.6. Measurement of Degree of Skin Laxity (DSL)

During the period of this study, mice backs were epilated with 6% (w/w) sodium sulfide 2 days before measuring degree of skin laxity each time. Briefly, the dorsal skin, about 1 cm away from the tail root, was gently stretched by left hand with mice hind limbs off the table top slightly. Right hand controls mouse movement by pulling tail. The stretch length was measured immediately when mice were immobile. The DSL was defined as the following equation: DSL (mm) = stretch length of dorsal skin.

2.7. Measurement of Spleen Index (SI) and Thymus Index (TI)

The mice were weighed and sacrificed. Spleen and thymus were excised from the mice and weighed immediately. The spleen index (SI) and thymus index (TI) were calculated according to the following equation: SI or TI (mg/g) = (weight of spleen or thymus)/body weight.

2.8. Histological Analysis

After eight weeks, mice were sacrificed and dorsal skin samples were dissected out immediately. 4 skin samples (About 1 cm2) in each group were fixed in 4% buffered neutral formalin solution for 24 h, and embedded in paraffin. Serial sections (7 μm) were put onto silane-coated slides and stained with haematoxylin–eosin (HE). The stained sections were further analyzed using an optical microscope. 1 representative image of HE-stained dorsal skin section in each group was presented in part of results.

2.9. Measurement of Moisture Content

Mice backs were epilated with 6% (w/w) sodium sulfide 2 days before sacrificing mice. Dorsal skins were collected after mice were sacrifice and skin moisture was measured immediately. The moisture content of skin sample was determined according to GB/T5009.3-2010, a national standard of China for measuring moisture content. This method was employed to measure moisture content of skin in several previous reports. Briefly, about 0.1 g of powdered skin sample was put into weighing bottle and dried in an oven at 105 °C for 4 h. The moisture content was calculated according to the following equation:

Moisture content = (m1 − m2)/(m1 − m3) × 100

m1, m2 and m3 is the weight of weighing bottle plus skin sample, weighting bottle plus dry finished skin sample and weighing bottle, respectively.

2.10. Determination of Hyaluronic Acid (HA) Content

About 0.1 g skin tissue was powdered in a liquid nitrogen bath and homogenized in pre-cooling saline. After centrifugation at 14,000× g for 15 min at 4 °C with a refrigerated centrifuge (TGL-185, Pingfan Co., Ltd., Changsha, China), the supernatant was collected to analyze the hyaluronic acid (HA) content using a commercial HA measurement kit (Nanjing Jiancheng Bio Inst., Nanjing, China).

2.11. Determination of Collagen Content

A commercial hydroxyproline assay kit (Nanjing Jiancheng Bio Inst., Nanjing, China) was used to analyze the Hyp content. Briefly, about 0.05 g skin tissue was totally hydrolyzed, oxidized and reacted with dimethyl-amino-benzaldehyde. The end product has a maximal absorption at 550 nm. The Hyp content in the skin was finally determined by comparison with the absorbance of the Hyp standard. The collagen content was calculated according to the Hyp content using a conversion factor of 8.00.

2.12. Ratio of Type I to Type III Collagen

Commercial type I and type III collagen assay kits (Nanjing Jiancheng Bio Inst., Nanjing, China) were used to analyze the relative content of type I and type III collagen. The ratio of type I to type III collagen was calculated according to the following equation: ratio of type I to type III collagen = content of type I collagen/content of type I collagen.

2.13. Antioxidant Indicators Analysis

Skin tissue were powdered in a liquid nitrogen bath and homogenized with 9 weights of pre-cooling saline. Homogenate was centrifuged at 14,000× g for 15 min at 4 °C with a refrigerated centrifuge (TGL-185, Pingfan Co., Ltd., Changsha, China) to collect the supernatants. Total protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Solarbio, Beijing, China). The SOD activity, CAT activity and malondialdehyde MDA content (expressed as MDA equivalents) were analyzed using the corresponding enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bio Inst., Nanjing, China) according to the manufacturer‘s instructions and the results were expressed in U/mg protein or nmol/mg protein.

2.14. Statistical Analysis

Results are expressed by the means ± standard deviation (SDs). Comparisons between two groups were analyzed by Student’s t-test. Differences between the means of the individual groups were analyzed using the analysis of variance (ANOVA) with Duncan’s multiple range tests. A difference was considered statistically significant when p < 0.05. All computations were performed with SPSS Statistics 19 (IBM, Chicago, IL, USA).

3. Results

3.1. Characterization of Collagen Peptides

Alcalase and collagenase (two optimized enzymes in our prior study) were used for producing different collagen peptides (named ACP and CCP, respectively). The molecular weight distributions of ACP and CCP. ACP and CCP had a similar molecular weight distribution. Both ACP and CCP mainly consisted of peptides in molecular weight ranges of < 500 Da (more than 50%), and the peptides of < 1000 Da accounted for approximately 70% and 74%, respectively.

The amino acid compositions of ACP and CCP. ACP and CCP had similar amino acid compositions. Gly is the most dominant amino acid in ACP and CCP, which is consistent with the Gly-X-Y repeating sequence in the collagen macromolecule. In addition, ACP and CCP are also rich in Pro, Glu, Phe, Arg and Thr.

3.2. Degree of Skin Laxity

As summarized, degree of skin laxity (DSL) of young (Y) group was increased during the experiment period but significantly lower than that of model group (old mice), which indicated that skin laxity was increased in an age-dependent manner. During the 8 weeks, the DSL of mice in CPs-treated groups (ACP and CCP groups) decreased over time compared with that in week 0. Significant differences in DSL were seen between ACP-400 group and time-matched model group at week 6 (p < 0.05), and the DSL of ACP-400 had no significant difference with that of young group. Furthermore, when the time of oral intake of ACP was as long as 8 weeks, the DSL of all ACP-fed groups decreased to the level of young group (p > 0.05), and some of groups (ACP-800 and CCP-400) were even better than the young group. Similarly, oral administration of proline at a dose of 400 mg/kg body weight also reduced the DSL with a significant difference observed compared with the time-matched model group at week 8 (p < 0.05).

3.3. Body Weight, Spleen Index (SI) and Thymus Index (TI)

The body weight of young group was increased during the experiment period, whereas that of model group remained stable. Treatment with ACP (200, 400 and 800 mg/kg body weight), CCP and proline (400 mg/kg body weight) for 8 weeks caused no statistically significant differences in the body weight compared with the untreated model group. Furthermore, the SI and TI of ACP groups, CCP and proline groups also had no significant difference compared to that of the model group. The body weight and organ indices could be measured to preliminarily determine whether a sample or sample dose had obvious toxicological effects on the animal subjects. There was no obvious atrophy, hyperplasia or swelling of spleen and thymus after CPs and proline intake. Based on these results, it was concluded that oral administration of CPs from bovine bone at doses of 200–800 mg/kg body weight, or proline at 400 mg/kg body weight for 8 weeks, had no obvious toxicological effects.

3.4. Skin Histology

The results of morphological examination of mice dorsal skin. Skin collagen fibers in dermis were stained a light red with haematoxylin–eosin (HE). In the model group (old mice), lighter red and more space (green arrow) were observed in the dermis tissue than were those of the young group. There were thinner dermis and less sebaceous gland (red arrow) in the model group compared with the young group. After ACP and CCP intake, the space in the dermis tissue was decreased and the fibers appeared to be denser and more organized compared with the model group. Besides, the number of sebaceous gland was increased when treated by ACP, especially at dose of 800 mg/kg body weight. These results indicated that ACP improved the aged collagen fibers in skin dermis in a dose-dependent manner. Similarly, the sparse, fragmented, and disorganized fibers were also obviously improved and the number of sebaceous gland was greatly increased by the oral administration of proline at a dose of 400 mg/kg body weight for 8 weeks.

3.5. Skin Components

The results of skin moisture, hyaluronic acid (HA), collagen content and ratio of type I to type III collagen. Skin moisture content and ratio of type I to type III collagen in the model group (old mice) were significantly lower than that in the young group (all p < 0.05), indicating that skin moisture content and ratio of type I to type III collagen were decreased with age. Skin HA and collagen contents were also lower than that in the young group, although there was no significant difference observed compared with the model group. Ingestion of CPs (both ACP and CCP) and proline had no significant effect on skin moisture and HA contents compared with the model group. In contrast, oral administration of ACP (200, 400 and 800 mg/kg body weight) caused a dose-dependent increase in the collagen content, and there was a significant difference in the collagen content between the group receiving 800 mg/kg of ACP and the model group (p < 0.05). Ingestion of CCP at a dose of 400 mg/kg body weight also increased the collagen content in skin (p < 0.05 vs. the model group). However, proline intake at a dose of 400 mg/kg body weight had no significant effect on collagen content compared with the model group. A dose-dependent increase was also observed for ratio of type I to type III collagen in the ACP-fed groups, and there were significant differences between the groups receiving 400 and 800 mg/kg of ACP and the model group (all p < 0.05); whereas CCP ingestion at a dose of 400 mg/kg body weight had no significant effect on ratio of type I to type III collagen compared to the model group. Oral administration of proline also significantly increased ratio of type I to type III collagen in skin (p < 0.05 vs. the model group).

3.6. Antioxidant Indicators

The superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content in skin. The SOD and CAT activities in the M group were significantly lower in the skin compared to the Y group (p < 0.05); whereas the MDA level in the model group was higher than that in the young group (p < 0.05). Oral administration of CPs (both ACP and CCP) significantly increased the SOD and CAT activities and reduced the MDA level (all p < 0.05 vs. the model group). Besides, the increase of SOD and CAT activities and the decrease of MDA level in ACP-fed groups showed a dose-dependent manner. In contrast, proline ingestion had no obvious significant effect on these three antioxidant indicators.

4. Discussion

Skin aging is consisted of chronological aging and photoaging. There are some difference in clinical signs and underlying mechanisms for these two processes. Collagen peptides (CPs) have been widely reported to exert beneficial effects on photoaging skin, but few studies was carried out to evaluate their effect on chronologically aged skin. In present study, 13-month-old Kunming mice, equivalent to 45 years old of human life, were employed to investigate the effect of CPs on chronologically aged skin. In several previous clinical trials, a daily dose of 2.5 g or 5 g of CPs has been employed in adult subjects, and these doses were considered to be safe. According to the conversion of animal doses to human equivalent dose (HED) based on the body surface area (BSA), about daily dose of 500 or 1000 mg/kg body weight could be used in mice. In addition, daily doses of 50–200 mg/kg body weight have also been used in several animal experiments. Therefore, doses of 200, 400 and 800 mg/kg body weight/day were employed in the present study.

Skin laxity is a main feature of natural skin aging and is increased with age. Therefore, skin laxity was dynamically evaluated by measuring the degree of skin laxity (DSL) to observe the effect of CPs intake on chronologically aged skin. An obvious beneficial effect was observed after 8 weeks of CPs intake. Therefore, mice were sacrificed and samples were collected for further treatment and analysis after 8 weeks. Unexpectedly, proline (abundant in collagen) ingestion also significantly improved skin laxity. These results provided guidance for the application of CPs or proline against chronological skin aging. The 8-week duration of CPs ingestion might be equivalent to several years old of human life in terms of life span. But it does not mean that the beneficial effects of CPs could be observed only after several years duration of CPs intake, because several studies have reported that significant beneficial effects of CPs on aging skin could be observed after 6 to 12 weeks in both clinical trials and animal experiments.

As the main component of the skin dermis, collagen has been reported to be beneficial in improving skin laxity and decreasing the appearance of wrinkles and its reduction in the quantity and quality is a major cause of laxity and wrinkles. In the chronologically aged skin, dermal collagen fiber became sparse, fragmented and disorganized. However, intake of CPs (both ACP and CCP) repaired collagen fibers and the fibers appeared to be denser and more organized compared to the aged skin. Collagen in skin mainly consists of type I and type III collagen. Collagen production and the ratio of type I to type III collagen is decreased gradually with age. Type I collagen tend to form broader bundles of fibers, while type III collagen forms narrow bundles. A decrease in the diameter and number of the collagen bundles is correlated with the decrease in load and tensile strength reported in aging skin. Oral administration of CPs increased the collagen content and ratio of type I to type III collagen in a dose-dependent manner, which suggested that CPs improved skin laxity by changing skin collagen quantitatively and qualitatively. In contrast, the skin moisture and hyaluronic acid (HA) were not affected by CPs ingestion. HA is a key molecule involved in skin moisture, because it has a unique capacity to bind and retain water molecules. Taken together with the results of the current study, it was concluded that oral administration of CPs had beneficial effect on chronologically aged skin by improving skin laxity, but it had no influence on moisture retention of skin.

Interestedly, ingestion of proline also had beneficial effect on chronologically aged skin in terms of skin laxity, collagen content and ratio of type I to type III collagen. This result of in vivo study was consistent with that of a previous in vitro experiment which demonstrated that proline could increase the collagen synthesis of confluent fibroblasts, but it did not stimulate the proliferation of fibroblast. Watanabe-Kamiyama and coworkers have reported that proline could reach the skin after proline intake. Therefore, we speculated that proline intake exerted beneficial effect on chronologically aged skin by increasing the collagen synthesis of skin fibroblasts.

It has been widely accepted that oxidative stress plays a critical role in initiating and driving the signaling events that result in skin aging. Study has reported that the production of reactive oxygen species (ROS) was increased in photoaged and chronologically aged skin. In addition to directly attacking macromolecules, such as proteins, lipids, DNA and RNA, the excessive ROS also initiate several signaling pathways, including mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and further activate transcription factor activator protein-1 (AP-1). AP-1 induces collagen degradation by upregulating collagen-degraded enzymes such as matrix metalloprotease (MMP)-1, MMP-3 and MMP-9 and downregulating the biosynthesis of collagen. These changes in the skin lead to the phenotype of aged skin. Therefore, antioxidants or free radical scavengers, such as ascorbic acid and polyphenols were reported to improve skin aging by scavenging excessive ROS. Normally, endogenous antioxidant enzymes are able to scavenge the excessive ROS to protect skin tissues from oxidative injuries. SOD and CAT are two antioxidant enzymes that inactivate superoxide anions and hydrogen peroxide, respectively. MDA is a product of lipid peroxidation and is usually quantified to estimate the lipid peroxidation extent induced by ROS. The SOD and CAT activities were decreased and MDA content was increased with age. However, CPs (ACP and CCP) intake could increase SOD and CAT activities and decrease MDA content, indicating that ingestion of collagen peptides from bovine bone had the ability to decrease ROS in skin. The decreased ROS in skin might help to increase the biosynthesis of collagen and decrease the collagen degradation by reducing the MMPs production. Indeed, several previous studies have reported that collagen hydrolysate ingestion could increases skin collagen expression and suppresses MMP-1 and MMP-2. In vitro study had demonstrated that ACP and CCP had high antioxidant capacity base on the hydroxyl radicals and ABTS + scavenging assays (data not shown). In addition, it has been widely reported that nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in regulating antioxidant enzymes against oxidative stress. Therefore, we speculate that CPs exerted their antioxidant effect in a direct and/or indirect manner. It is also possible that CPs exerted their beneficial effects on chronological aged skin in other ways, as previous studies have reported that Pro–Hyp in human blood after oral ingestion of CPs stimulates fibroblast growth. It should be noted that proline intake did not have an obvious effect on skin antioxidant capacity. These results suggested that CPs had more complex action mechanisms underlying anti-aging effect than proline.

Bovine bone is an abundant source of gelatin. The CPs from bovine bone is mainly concentrated on its beneficial effect on bone metabolism. However, the current study found that the CPs from bovine bone also had beneficial effect on skin aging. The CPs prepared in this study mainly consist of oligopeptides (< 1000 Da). It was reported that small peptides, especially the di-and tripeptides, are more easily absorbed in the intestinal tract than larger molecules, and oligopeptides are more bioactive than proteins, polypeptides and free amino acids. Therefore, we speculated that ACP and CCP are readily absorbed and might exhibit potential biological effects once they are orally administered. Another purpose of this study is to preliminary investigate whether different CPs prepared by Alcalase and collagenase have different effects on chronologically aged skin. Based on the present results, it can be drawn that the beneficial effects of ACP were slightly better than those of CCP. Therefore, Alcalase is a favorable enzyme to produce CPs with beneficial effects on skin aging in food and medical industries. The present result of molecular weight distribution provides a guide for testing and controlling the quality of CPs. Besides, CPs should be protected from oxygen and light because of its easy oxidation.

5. Conclusions

In summary, the present study demonstrated oral administration of collagen peptides from bovine bone could improve the laxity of chronologically aged skin by increasing skin collagen content and ratio of type I to type III collagen, but it had no effect on moisture retention of skin. The beneficial effects of collagen peptides prepared by Alcalase (ACP) were slightly better than those of collagen peptides prepared by collagenase (CCP). Another action mechanism underlying the beneficial effects on aged skin of collagen peptides may be involved in increasing the antioxidant properties in the body. Proline intake also improved the laxity of chronologically aged skin but it did not affect the skin antioxidant capacity. These results suggest that collagen peptides from bovine bone and proline are potential dietary supplements for use against skin aging in chronologically aged process.

Acknowledgments

This study was supported by the earmarked fund from China Agriculture Research System (CARS-46) and National Natural Science Foundation of China (NSFC, No. 31271846).

Author Contributions

Bo Li conceived and designed the experiments; Hongdong Song, Siqi Zhang and Ling Zhang performed the experiments and analyzed the data; Hongdong Song and Bo Li wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.

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Abstract

BACKGROUND: Skin dryness and an accelerated fragmentation of the collagen network in the dermis are hallmarks of skin aging. Nutrition is a key factor influencing skin health and consequently its appearance. A wide range of dietary supplements is offered to improve skin health. Collagen peptides are used as a bioactive ingredient in nutricosmetic products and have been shown in preclinical studies to improve skin barrier function, to induce the synthesis of collagen and hyaluronic acid, and to promote fibroblast growth and migration. Our aim was to investigate the effect of oral supplementation with specific collagen peptides on skin hydration and the dermal collagen network in a clinical setting.

METHODS: Two placebo-controlled clinical trials were run to assess the effect of a daily oral supplementation with collagen peptides on skin hydration by corneometry, on collagen density by high-resolution ultrasound and on collagen fragmentation by reflectance confocal microscopy. Human skin explants were used to study extracellular matrix components in the presence of collagen peptides ex vivo.

RESULTS: Oral collagen peptide supplementation significantly increased skin hydration after 8 weeks of intake. The collagen density in the dermis significantly increased and the fragmentation of the dermal collagen network significantly decreased already after 4 weeks of supplementation. Both effects persisted after 12 weeks. Ex vivo experiments demonstrated that collagen peptides induce collagen as well as glycosaminoglycan production, offering a mechanistic explanation for the observed clinical effects.

CONCLUSION: The oral supplementation with collagen peptides is efficacious to improve hallmarks of skin aging.

© 2015 The Authors. Journal of Cosmetic Dermatology Published by Wiley Periodicals, Inc.

 

Abstract

Nutraceuticals containing collagen peptides, vitamins, minerals and antioxidants are innovative functional food supplements that have been clinically shown to have positive effects on skin hydration and elasticity in vivo. In this study, we investigated the interactions between collagen peptides (0.3–8 kDa) and other constituents present in liquid collagen-based nutraceuticals on normal primary dermal fibroblast function in a novel, physiologically relevant, cell culture model crowded with macromolecular dextran sulphate. Collagen peptides significantly increased fibroblast elastin synthesis, while significantly inhibiting release of MMP-1 and MMP-3 and elastin degradation. The positive effects of the collagen peptides on these responses and on fibroblast proliferation were enhanced in the presence of the antioxidant constituents of the products. These data provide a scientific, cell-based, rationale for the positive effects of these collagen-based nutraceutical supplements on skin properties, suggesting that enhanced formation of stable dermal fibroblast-derived extracellular matrices may follow their oral consumption.

Introduction

The biophysical properties of the skin are determined by the interactions between cells, cytokines and growth factors within a network of extracellular matrix (ECM) proteins1. The fibril-forming collagen type I is the predominant collagen in the skin where it accounts for 90% of the total and plays a major role in structural organisation, integrity and strength2. A complex network of interlaced collagen fibrils in the dermis provides support to the epidermis, and together with elastin and microfibrils gives the skin its elasticity and resilience1. In addition, proteoglycans and polymeric oligosaccharides, including abundant hyaluronic acid, play a key role in skin hydration.

Collagen I, elastin and proteoglycans, the three major groups of dermal ECM proteins, are secreted mainly by dermal fibroblasts activated by TGFβ, a multifunctional growth factor regulating the expression, deposition and turnover of skin extracellular matrix proteins1. Production of collagen and of the other components of the extracellular matrix is high when there is a sufficient level of mechanical tension on fibroblasts. When this tension is reduced, for example with age, the production of the matrix proteins falls and there is an increase of matrix-degrading enzymes3. The mature interstitial collagen fibrils are resistant to most proteolytic enzymes, but are susceptible to degradation by the collagenolytic matrix metalloproteinases MMP-1, MMP-8 and MMP-134. Elastolytic MMPs include the macrophage metalloelastase MMP-12 and the weakly elastolytic MMP-3 which is expressed by fibroblasts.

The skin is subject to intrinsic (chronological) and extrinsic (environmental and lifestyle factors including UV radiation and smoking) ageing, which are both associated with histopathological and immunohistochemical changes5. Intrinsic ageing is characterised by cell senescence6, and altered levels of collagen7, elastin8 and glycosaminoglycans, including hyaluronic acid9. In extrinsic ageing, there is loss of reticular collagen and an accumulation of disorganised elastic fibres and glycosaminoglycans. Photo-aged skin (UV-irradiated) displays alterations of the extracellular matrix, with an increase in the expression of matrix metalloproteinases and collagenases10. Increased expression and activity of MMPs, notably MMP-1, MMP-3 and MMP-911,12, has been associated with photo-ageing, and a direct effect of UV on the integrity of elastic microfibril associated proteins and on the elastin network has been suggested8,13.

A major cause of ageing-related skin damage is thought to be a consequence of decreased antioxidant defences leading to increased levels of intracellular reactive oxygen species (ROS). These form through aerobic metabolism and stimulate signal transduction resulting in the increased expression of MMPs and decreased collagen I synthesis14. The generation of ROS has been directly associated with protein damage, and with up-regulation in the expression and activity of MMPs in intrinsically and extrinsically aged skin15,16. Moreover, the ECM proteins can work as skin photo-sensitizers, enhancing the genotoxicity of a given dose of UV irradiation, contributing therefore to skin photo-ageing17. With age, the imbalance between synthesis and degradation of the ECM proteins leads to glycation and the consequent formation and accumulation of AGEs (advanced glycation end-products), which are a hallmark of age-related diseases18. Further, with age, the ability to replenish collagen naturally decreases by about 1% per year19. Thus, the administration of antioxidants might help in counteracting ROS-induced signs of ageing20. Along this line, it was shown that the administration of antioxidants can decrease oxidative stress in a model of prematurely ageing mice21. Moreover, clinical studies have shown that the oral administration of antioxidants can help improve skin condition in photo-aged skin22,23 and UV-induced erythema24.

In addition, evidence from placebo-controlled clinical studies supports the notion that daily oral consumption of collagen peptides derived by hydrolysis of native porcine and piscine collagen improves the density and integrity of the collagen network, hydration and elastic properties of normal skin25,26. Further, dietary supplements combining piscine collagen peptides with other active ingredients including hyaluronic acid, antioxidants, vitamins and minerals improve the appearance of the ageing skin27,28,29. However, the cellular mechanisms underpinning these observations remain to be elucidated.

The aim of this in vitro study is to investigate the effects on normal human dermal fibroblast synthesis of collagen I and elastin, release of transforming growth factor-β (TGF-β), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinases (MMP), MMP-1 and MMP-3, and elastin degradation of collagen bioactive peptides, alone and in combination with other bioactive compounds found in two different collagen-based nutraceutical supplements previously reported to increase skin elasticity in vivo28,29.

Our hypothesis is that the antioxidant activity associated with the additives within these nutraceutical products interacts with the effects of the collagen peptides to enhance the stability of matrix proteins by inhibiting release of MMP-1 and MMP-3 in dermal fibroblast culture.

Materials and Methods

Test products

The collagen peptides, natural antioxidants and other bioactive molecules tested in this study are those found in the collagen-based nutraceutical supplements ACTIVE GOLD COLLAGEN® (ACTIVE) and GOLD COLLAGEN® FORTE (FORTE), which are manufactured by Minerva Research Labs (London, UK). The collagen peptides component (0.3–8 kDa; Peptan® by Rousselot) was tested on normal primary human dermal fibroblasts in culture in the absence and presence of a full combination of other active ingredients at the concentrations shown in Table 1. These data are presented in the main results section. The collagen peptides were also tested following addition of individual active ingredients in the combinations shown in Table 1 to investigate possible additive effects of individual components. These data are described in the main results section and are presented graphically in Supplementary Figs S3–S8.

Collagen Peptides Production

Peptan® collagen peptides are produced by hot water extraction of the endogenous collagen from fish skin, filtration, concentration, subsequent standardized and controlled enzymatic hydrolysis, sterilization and spray-drying. The production follows GMP guidelines and is HAACP-controlled in a IFS and ISO certified plant.

Molecular Weight distribution

The molecular weight of Peptan® collagen peptides is between 0.3 and 8 KDa. The molecular weight distribution of these collagen peptides is determined by high performance size exclusion chromatography (HPSEC) using an Agilent HPLC, 1260 Infinity series (G1316A, G1329B, G1311C, G1315D) with a TSKgel SWXL precolumn und a G2000SWXL column (Tosoh Bioscience). Analysis is performed with the WinGPC software (PSS). Samples are eluted from the column with 170 mM phosphate buffer containing 15% acetonitril and monitored with UV detection. Calibration is performed with the Narrow Calibration Standard (Low FILK).

Cell Culture

Normal adult human dermal fibroblasts (NHDF) were purchased from Lonza (Walkersville, MD, USA) and grown in fibroblast growth medium-2 (FGM), containing 2% FBS (Lonza), at 37 °C and 5% CO2. Cells at passage 3–7 were seeded into 24 and 96 well plates at a density of 5 × 104 cells/well, and 5 × 103 cells/well for protein analysis and proliferation assays respectively. The fibroblasts were grown for 16 hours, quiesced for 24 h in medium containing 0.3% FBS and then treated for 48 hours with combinations of the bioactive constituents of ACTIVE or FORTE (Table 1) at a concentration found in a 1:50 dilution of the whole product. The rationale for testing components at this dilution is based on the absorption and distribution of avian collagen peptides of similar composition in an animal model, as detailed in the Supplementary Material.

These treatments were added in FGM containing 0.3% FBS, supplemented with 100 µM L-ascorbic acid and 100 µg/ml > 500 kDa dextran sulphate (Sigma-Aldrich, St. Louis, USA), which will be referred to hereafter as ‘crowded medium’30,31. Under these conditions cells remained viable, as confirmed by their adherence to cell culture plates and proliferation over 48 hours, as described in section 2.7. In all experiments, cell culture supernatants were collected, cleared by centrifugation at 1000 g for 10 minutes (min) at 4 °C and stored at −80 °C until assayed.

Amino Acids composition

The samples were hydrolysed in 9 M HCl at 110 C for 20 h, and subsequently derivatized and stabilized by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Fluor reagent kit WAT052880). These fluorescent derivatives were separated by RP-HPLC on a Waters 2695 Alliance HPLC Separation Module and detected by a fluorescent detector. Quantification is performed by the Software Waters – Empower 3 using standards for each amino acid (Amino Acid Standard H WAT088122). Peptan® collagen peptides are characterised by a high content of Glycine, Hydroxyproline/Proline and Glutamic Acid which represent 56% of the total amino acids. These collagen peptides comprise also Arginine (8%), Alanine (8%), essential amino acids (16%) and other amino acids (12%).

Collagen I

Cells were washed twice with phosphate buffered saline. Cell-associated collagen was solubilised by the addition of 0.5 M acetic acid (250 µl/well) and gentle agitation at 4 °C for 32 hours, before adding 0.1 mg/ml pepsin from porcine mucosa (Sigma-Aldrich) and continuing agitation for a further 16 hours. Pepsin digestion was then inhibited with 2 µg/ml pepstatin A (Sigma-Aldrich). Cell samples were stored at -80 °C until assayed. A novel quantitative immuno-blot assay for native collagen I was developed and validated by Western blot analysis of TGFβ-activated NHDF cells and supernatants (Supplementary Fig. S1). Standards (0.625–10 ng calf skin collagen I, Sigma Aldrich) and samples (100 µl) diluted with PBS (acid-soluble cell fraction, 1:10, and supernatants, 1:20), were loaded onto nitrocellulose membrane using a vacuum manifold to create protein dots. Blots were dried, blocked with 5% dried skimmed milk powder and 2% Tween 20 in PBS and incubated overnight at 4 °C with rabbit anti collagen I antibody (Abcam, Cambridge, UK) at 200 ng/ml in block buffer. The blots were then incubated with 50 ng/ml goat anti rabbit-HRP antibody (Dako, Glostrup, Denmark) for 1 hour at room temperature. Blots were visualised using chemiluminescence (Thermo-Fisher, Waltham, USA).

Elastin

Cells were lifted with trypsin-EDTA (Sigma-Aldrich) and diluted 3:1 with 1 M oxalic acid to 0.25 M oxalic acid. Cell samples were incubated at 95–100 °C for 2 hours, with intermittent mixing, and the entire cell and supernatant samples from each well were assayed separately for solubilised elastin using the Fastin elastin assay kit (Biocolor, Carrickfergus, UK), following manufacturer’s instructions.

AP-1 analysis

NHDF were lysed in 20 mM TRIS-HCl, pH 7.6, containing 150 mM NaCl plus 1% Triton X-100, lysis buffer and 2 × protease inhibitors, protease cocktail I (Calbiochem), and Complete® protease inhibitor (Roche), PhosSTOP® phosphatase inhibitor (Roche), 4 mM MgCl2 and benzonase (Sigma-Aldrich) at 1:1000 dilution in sample buffer (35 ul per well of 24-well plate).

Samples were mixed with sample buffer and separated on 10% SDS-PAGE and proteins transferred to nitrocellulose using semi-dry electrophoresis. Membranes were blocked with 3% dried skimmed milk powder in TRIS (20 mM) buffered saline (150 mM) plus 0.1% Tween-20, and stained with rabbit anti-p-c-jun (Ser 63/73) at 200 ng/ml, and secondary goat-anti-rabbit-HRP (Sigma-Aldrich) at 1:2000 dilution. Chemiluminescence (Promega) was used to detect bands following 30 mins exposure in the ChemiDoc imager (BioRad).

ELISAs

Cells were lysed using hypotonic lysis buffer (1% Triton X-100 in 10 mM Tris-HCl buffer, pH 7.4) containing 2 × protease cocktail I inhibitors (Merck (Calbiochem), Darmstadt, Germany) and stored at −80 °C until assayed. Supernatants were analysed neat for TGF-β, PAI-1 and desmosine, and at a 1:25 dilution for MMPs -1 and-3. Total TGF-β, total MMP-1, total MMP-3, TIMP-1 and PAI-1 ELISA kits from R&D systems (Abingdon, UK) were used according to the manufacturer’s instructions.

A desmosine ELISA was performed using an in-house competitive ELISA as previously described32. In brief, the wells of a 96-well Nunc maxisorp microtitre plate were coated with 250 ng desmosine-egg-albumin complex (Elastin Products Company (EPC), Missouri, USA) in 100 µl bicarbonate buffer, pH 9.6 (Sigma-Aldrich), overnight at 4 °C. Desmosine standards (EPC) were prepared in the range 0–2000 ng/ml in 100 mM Tris-HCl pH 7.2, containing 0.1%Tween-20. Standards and samples (100 µl) were added to 200 µl of a 1:3000 rabbit anti-desmosine serum (EPC) and incubated at 37 °C for 30 min. Plates were washed, and standards and samples (100 µl in duplicate) were added to the wells and incubated for 2 h at 4 °C. Biotinylated swine anti-rabbit (Dako) antibody, 100 µl at 1:1000 dilution, was added to each well and incubated for 1 h at room temperature. Streptavidin-HRP complexes (Vector Labs), 100 µl at 1:1000 dilution, were added for 30 min at room temperature, followed by substrate (5.5 mM o-phenylene-diamine solution in TRIS-citrate buffer pH 6). Reactions were stopped by the addition of 100 ul 2 M H2SO4 and the plate read at 490 nm.

Cell Proliferation

The proliferation assay was performed after 48 hours of cell culture, as described in section 2.2, using the CyQuant NF assay kit (Thermo Fisher) according to the manufacturer’s instructions. A standard curve to calculate cell number was prepared with 100–50,000 cells per well, in triplicate, which were allowed to adhere for 4 h, then stained with CyQuant dye binding solution for 40 min, and fluorescence was measured at excitation/emission wavelengths 485/530.

Antioxidant activity

The antioxidant activity of the collagen peptides and the other bioactive ingredients tested was measured using a pyranine-based procedure to evaluate the total peroxyl scavenging capacity33,34. The method is based on the ability of antioxidants to prevent the bleaching of pyranine (200 μM) by peroxyl radicals generated from AAPH (2,2′-azo-bis- (2-amidinopropane) hydrochloride (200 mM). Trolox, a water-soluble analogue of vitamin E was used as a standard and values were expressed as Trolox equivalents (TE). Samples (25 μl) were mixed with 25 μl pyranine and incubated for 3 min at 37 °C. AAPH (50 μl) was added and the reaction monitored at 454 nm every minute for 80 min. The lag phase to bleaching was determined for samples and Trolox standards in the range 0 to 0.5 mM. Reagents were all from Sigma-Aldrich.

Statistical analysis

Fibroblast responses are presented as mean ± sem and were analysed by one-way ANOVA followed by Fisher’s LSD post-hoc test using GraphPad Prism, version 7, software. Correlation between the cumulative antioxidant activity of individual bioactives and elastin, MMP-1 and MMP-3 levels in NHDF cultures was analysed by one-tailed Pearson correlation coefficient test. Differences where p < 0.05 were considered to be statistically significant.

Results

It has previously been reported that TGF-β at 5 ng/ml strongly stimulates collagen synthesis by embryonic pulmonary fibroblasts in crowded culture30. Therefore, because TGF-β controls expression, deposition and turnover of collagens and other extracellular matrix proteins in the skin1, and primary dermal fibroblast responses to TGF-β as a positive control in cultures crowded with high molecular weight dextran sulphate have not previously been reported, we initially tested the effect of TGF-β (5 ng/ml) on NHDF protein synthesis and proliferation after 48 hour incubation under similarly crowded conditions, compared to media alone (Fig. 1). The values for baseline concentrations of all proteins in quiescent cultures are presented in the legend to Fig. 1. Significantly more collagen I was found in the supernatant (2.5 ± 0.6 ng/well) compared to the cell layer (0.6 ± 0.2 ng/well). These values were normalised to 100% to test the relative effect of TGF-β. TGF-β stimulated a significant increase in collagen I in the cell lysate (942.0 ± 268.3%; Fig. 1a) and supernatant (154.6 ± 20.7%; Fig. 1b) compared to media alone (100%). Unlike collagen I, significantly more elastin was found, in quiescent cultures, associated with cell layers (11.4 ± 1.9 µg/well) compared to supernatants (6.5 ± 0.8 µg/well). Further, TGF-β significantly increased elastin in the cell lysate (165.1 ± 35.1%; Fig. 1d) and supernatant (151.8 ± 5.5%; Fig. 1e). TGF-β significantly decreased total MMP-1 (42 ± 7.4%; Fig. 1c), total MMP-3 (58.1 ± 9.9%; Fig. 1f) and desmosine (75.4 ± 4.1%; Fig. 1h) in the culture supernatants, significantly increased PAI-1 (721.2 ± 96.5%; Fig. 1g) in the supernatant and significantly increased the proliferation of NHDF (121.0 ± 5.4%; Fig. 1i) compared to the effect of media alone, normalised to 100%.

The effects of the addition of collagen peptides alone (C) and in combination with all the other bioactive and antioxidant constituents (All) present within the two nutritional supplements, ACTIVE and FORTE, at the concentrations described in Table 1, were tested and compared to the effect of media alone (designated as the 100% value in all experiments). In parallel, the effect of adding individual constituents to the collagen peptides, in the combinations described in Table 1, were tested and these results are presented in Supplementary Figs S3–S8. Collagen I, elastin and TGF-β synthesis by NHDF was measured in both the cell lysate and supernatant. PAI-1, MMP-1, MMP-3, TIMP-1 and desmosine were measured in the supernatant alone where they were most abundant.

Collagen I was found predominantly in soluble form when cells were grown in media alone (Fig. 2, legend). Although collagen peptides alone had no significant effect on collagen I in the supernatant, soluble collagen I was significantly increased in response to the combination of collagen peptides with the other five ACTIVE constituents tested (All; 265.8 ± 169% of media control; Fig. 2b). Similarly, although cell-associated collagen in the cell lysate was not significantly increased in response to collagen peptides alone (C; 142.5 ± 32.2%), in combination with all other ACTIVE constituents cell lysate collagen was significantly increased (All; 196.2 ± 16.2%; Fig. 2a). Considering the individual combinations of ACTIVE constituents (Table 1, Fig. Supplementary Fig. S3a) all those including glucosamine in combination with hyaluronic acid and collagen peptides, but not hyaluronic acid or collagen peptides alone, significantly increased cell-associated collagen I (Supplementary Fig. S3a,c). In contrast, the combination of collagen peptides with the other nine constituents of FORTE (All) did not increase cell-associated or soluble collagen I (Fig. 2c,d).

Unlike collagen, elastin was found predominantly in cell lysates in unstimulated cultures (Fig. 3, legend). The addition of collagen peptides alone (C) significantly increased the amount of soluble elastin (Fig. 3b,d). Moreover, in combination with the nine other bioactive constituents of FORTE (All) the effect was further significantly enhanced (Fig. 3d).

Although all combinations (Table 1, Supplementary Fig. S4c,d) of the FORTE and ACTIVE constituents increased soluble elastin in the supernatant compared to media alone, only FORTE constituents stimulated a further significant increase in soluble elastin compared to the collagen peptides alone (Supplementary Fig. S4d). However, there was no significant increase in the cell-associated elastin in cell lysates on addition of collagen peptides to any combination with other bioactives (Fig. 3a,c, Supplementary Fig. S4a,b).

Addition to dermal fibroblast cultures of the whole ACTIVE and FORTE products (including acidity regulators, stabilisers, natural sweeteners, flavourings and vitamins) at 1:50 dilution, to match the tested concentrations of individual components, significantly increased soluble elastin to 173 ± 27.9% and 187 ± 40%, respectively, of the media control value.

We first considered whether the observed increases in collagen and elastin concentrations were due to an autocrine effect of TGF-β released by dermal fibroblasts under specific culture conditions. However, TGF-β was detected at low (pg/ml) levels (Fig. 4, legend) and found in an inactive form (i.e. requiring acid activation for detection). TGF-β was significantly increased in cell lysates (All; 184.2 ± 32%; Fig. 4a) and in supernatants (All; 119.6 ± 7%; Fig. 4b) in response to collagen peptides in combination with all ACTIVE constituents. However, there was no significant increase in TGF-β in either the supernatant (Fig. 4d) or cell lysate (Fig. 4c) in response to addition of the constituents of FORTE.

Active TGF-β is a potent inducer of PAI-1 in NHDF culture supernatants (Fig. 1g). However, PAI-1 was not increased under any culture condition (data not shown), confirming that TGF-β was present only at low levels and in an inactive form. Further, while TGF-β strongly induced AP-1 activation, detected as an increase in phospho-c-jun in cell lysates (Supplementary Fig. S2) the collagen peptides and other bioactives under investigation did not (data not shown).

Because the measured increases in collagen and elastin potentially reflect reduced degradation by MMP-1 and MMP-3, respectively, we measured levels of these proteases and their cognate inhibitor, TIMP-1, in culture supernatants. A highly significant decrease in MMP-1 protein levels in supernatants (Fig. 5a,b) was seen in response to collagen peptides alone and the combination of all six ACTIVE constituents (All; 33.8 ± 3% of media control; Fig. 5a) and all ten FORTE constituents (All; 47.4 ± 7% of media control; Fig. 5b). The effect of the collagen peptides was further significantly increased on addition of the other constituents of FORTE (Fig. 5b). In addition, all combinations of ACTIVE and FORTE constituents significantly decreased MMP-1 levels in the supernatants (Supplementary Fig. S6a,b), but an additive effect of the FORTE constituents stimulated further significant decrease in MMP-1 protein levels compared to collagen peptides alone (Supplementary Fig. S6b).

Similarly, MMP-3 (Fig. 5c,d) was significantly decreased in supernatants in response to collagen peptides alone and in the presence of the six ACTIVE ingredients (All; 59.6 ± 18% of media control) and ten FORTE ingredients (All; 52 ± 14.8% of media control). The antioxidants and bioactive constituents of FORTE further significantly enhanced the effect of the collagen peptides (Fig. 5d). In fact, collagen peptides and all combinations of ACTIVE and FORTE constituents significantly reduced MMP-3 levels in supernatants (Supplementary Fig. S6c,d), but an additive effect of the FORTE constituents stimulated further significant decrease in MMP-3 protein levels compared to collagen peptides alone (Supplementary Fig. S6d).

In parallel with the significant decrease in MMP-1 and MMP-3 expression, a small but non-significant increase in TIMP-1 levels were observed (Fig. 5e,f) with collagen peptides alone that was not altered by addition of other constituents of ACTIVE or FORTE supplements.

In parallel with the decrease in MMP-3 concentrations, desmosine as a marker of elastin breakdown (Fig. 5g,h) was significantly decreased in response to collagen peptides in combination with all six ACTIVE constituents (All; 72 ± 12.6%; Fig. 5g), and all ten FORTE constituents (All; 64.3 ± 8.2%; Fig. 5h). All combinations (Supplementary Fig. S7a,b) of ACTIVE constituents, but only some combinations of FORTE constituents caused a significant decrease in the breakdown of elastin.

In view of the lack of evidence for autocrine effects of endogenous TGF-β in the cultures, we considered whether the observed inhibition of MMP-1 and MMP-3 expression was related to the total antioxidant activity of the added bioactives. In particular, the individual constituents of FORTE appeared to have an additive effect (Supplementary Fig. S6), and when added all together they significantly enhanced the effect of the collagen peptides (Fig. 5,b,d and h). The antioxidant activity of the collagen peptides and each of the bioactives under investigation was measured as peroxyl scavenging activity, and expressed as Trolox equivalents (Table 1). The data in Fig. 6 show the strong, significant, negative correlations between the cumulative antioxidant activity of the added FORTE constituents and MMP-1 and MMP-3 expression levels (Fig. 6c,d). Further, there is a significant negative correlation of soluble elastin with MMP-3 activity (Fig. 6a) and a significant positive correlation with antioxidant activity (Fig. 6b), indicating that basal expression and release of MMPs is driven by reactive oxygen species and that antioxidant activity added to the cultures was responsible for the further significant inhibition of MMP-3 release (Fig. 5d) and increase in soluble elastin (Fig. 3d, Supplementary Fig. S6d) beyond that seen with the collagen peptides alone.

Although there was no significant effect on proliferation (Fig. 7) when adding the collagen peptides alone, the combination of all ten constituents of FORTE significantly stimulated NHDF proliferation over 48 hours (All; 123.8 ± 6.5%; Fig. 7b). In addition, incubation of NHDF with HA, a constituent of ACTIVE (Supplementary Fig. S8a), and carnosine, a constituent of FORTE (Supplementary Fig. S8b) resulted in significantly increased cell proliferation that was not further enhanced by the addition of other bioactives.

Discussion

TGF-β is a well-recognised pro-fibrotic growth factor that promotes the deposition of ECM proteins, whilst limiting their degradation. The pro-fibrotic effects of TGF-β were confirmed in our model in which normal human dermal fibroblasts were cultured under conditions of macromolecular crowding to mimic the extracellular matrix environment of the skin. Exogenously added TGF-β1 significantly increased fibroblast proliferation, collagen and elastin synthesis and release of PAI-1, while inhibiting release and MMP-1 and MMP-3 and the breakdown of elastin. These effects are mediated by SMAD and AP-1 dependent signalling pathways that stimulate synthesis of collagen-I via Smad3 and PAI-1 via AP-135 and interact to repress MMP-1 and MMP-3 gene expression36,37. Redox signalling also plays an important role in the pro-fibrotic effects of TGF-β38.

Media crowded with macromolecules such as high molecular weight dextran sulphate (0.01% w/v) was reported to increase collagen synthesis by embryonic pulmonary fibroblasts 20–30 fold, compared to normal media31 and soluble procollagen was completely processed into insoluble collagen by dermal fibroblasts39, although the collagen was deposited as aggregates and not fibrils30. Using a sensitive in-house immunoblot method to detect collagen I, the presence of soluble collagen I indicated processing was incomplete in our study. However, it is possible that the high assay sensitivity detected more soluble collagen I than previous studies.

Collagen peptides significantly increased cell-associated collagen, but only in the presence of glucosamine (Supplementary Fig. S3). Positive effects on collagen levels may be associated with the ability of glucosamine to stabilise collagen matrices by reducing MMP synthesis, as demonstrated in synovial fibroblasts40. Although we found no peroxyl scavenging activity associated with glucosamine (data not shown), superoxide/hydroxyl-radical scavenging antioxidant activity of glucosamine hydrochloride has previously been reported, and it was suggested that glucosamine hydrochloride could be effectively employed as an ingredient in functional food, to alleviate oxidative stress41. However, the lack of any significant effect of adding antioxidants such as resveratrol, CoQ10, acai berry, lycopene and pomegranate on collagen expression, despite significant inhibition of MMP-1 expression, indicates an effect of glucosamine on collagen synthesis that is not mediated through antioxidant activity.

In contrast, soluble elastin was significantly increased by collagen peptides alone and with all combinations of antioxidants and other bioactives tested. Each of the constituent antioxidants found in FORTE appeared to have an additive effect on total soluble elastin synthesis. Moreover, when added to fibroblast cultures as the whole product (diluted 1:50 to give the same concentrations of the tested individual components), both collagen-based nutraceuticals significantly (p < 0.05) increased soluble elastin almost two-fold. This increase in elastin expression reflects what has already been observed in vivo. A significant increase in skin firmness (Young’s elasticity) was demonstrated in vivo after 90 days of supplementation with both whole collagen-based supplements investigated in this study28,29. Low molecular weight avian collagen peptides of similar composition, when orally administered in rats, were shown to accumulate preferentially in the skin42, at concentrations which could be scaled to the concentration of peptides consumed daily in the test products (100 mg/ml) and used in our in vitro study (2 mg/ml). Thus our in vitro observations support those from our previous in vivo studies, validating the model we have used to investigate dermal fibroblast responses to individual components of the products.

The very low levels of TGF-β released (25–50 pg/ml) by dermal fibroblasts in this model, and the lack of effect of any of the additives on the levels of active TGF-β, indicated that an autocrine effect of TGF-β was unlikely, and this is evidenced by the lack of effect on AP-1 activation and PAI-1 expression.

Collagen peptides alone and in combination with antioxidants and other bioactives significantly reduced MMP-1 and MMP-3 expression, in the absence of any change in TIMP-1 levels. Notably, the constituents of FORTE appeared to have an additive effect on inhibition of MMPs expression, as was also seen for the increase in soluble elastin in culture supernatants. A major cause of ageing-related skin damage is thought to be due to increased levels of ROS and oxidative stress14 which directly and indirectly, through increased expression of MMPs, damage structural proteins43. MMPs -1, -2, -3 and -9 are mainly responsible for ECM damage and degradation, and can fully degrade collagen together. However, the only MMP that can damage intact collagen I fibres is MMP-1, while MMP-3 is capable of degrading elastin43. Protective effects of the collagen peptides and other constituents on elastin integrity are indicated by a decrease in desmosine, a cross-linked amino acid and specific marker of elastin breakdown, in the cultures. The negative correlation of soluble elastin concentration with MMP-3 supports the notion that MMP-3 is responsible for elastin breakdown and the generation of desmosine in the cultures.

The significant positive association between total peroxyl scavenging activity of the additives and soluble elastin content in the cultures may now suggest a mechanistic role for antioxidant activity in the observed increase in soluble elastin. Potentially the effect is mediated by increased elastin transcription in the presence of the antioxidants, as previously shown for the effect of the antioxidant N-acetylcysteine44 or decreased expression of MMP-1 and MMP-3, which was previously reported for the effect of the antioxidant Tiron45 and mitochondria-targeted vitamin E46 on dermal fibroblasts. The significant negative correlation of the MMPs with antioxidant activity indicates that the antioxidants are protecting the cells from constitutive intracellular ROS, activation of AP-1 signalling and MMP expression14. These effects transcend any potential activities associated with the physiological (100 µM) concentration of ascorbic acid added to the culture medium as a co-factor for prolyl and lysyl hydroxylases involved in collagen synthesis, since 88–98% of ascorbate disappears from culture medium within 24 h47 and we found the peroxyl scavenging activity of 100 µM ascorbic acid was low (6 µM TE).

Finally, collagen peptides in combination with antioxidants and other bioactives under investigation, but not alone, stimulated fibroblast proliferation, and the magnitude of the effect was similar to that seen with TGF-β. A strong effect of HA on dermal fibroblast proliferation was not further increased by other additions. While HA has not been shown to be directly mitogenic48, through facilitating proliferation in response to other mitogenic factors such as TGF-β49 and, in this instance, collagen peptides, HA may have an important but indirect role in cell proliferation. Similarly, the stimulating effect due to addition of carnosine on fibroblast proliferation was not further enhanced by the other additions. McFarland and Holliday (1999) showed in their classical experiments that carnosine enhances the proliferative potential of fibroblasts by protecting the cells from telomere shortening50. Together with stimulating effects on dermal fibroblast proliferation51 carnosine may play an important role in skin regeneration.

In conclusion, 48 hour incubation of dermal fibroblasts with collagen-derived peptides and other nutraceutical constituents increases structural ECM protein synthesis, particularly elastin, and decreases synthesis of MMPs -1 and -3 and the elastin degradation product desmosine. The effect of these collagen-based supplements and their constituents therefore support normal adult human dermal fibroblast function in terms of potentially increasing matrix stability in the skin. This effect is not likely to be mediated through TGF-β signalling pathways but is more likely correlated with antioxidant effects on dermal fibroblast MMP expression.

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